The long-term goals of this proposal are to define heme transport and recycling in iron metabolism. Iron deficiency is the most common nutritional disorder in the world. The most common clinical manifestation of iron deficiency is anemia due to impaired hemoglobinization of red blood cells (RBCs). This is because over 70% of iron in the human body is used as heme in hemoglobin production. In healthy adults, about 90% of body iron is derived from recycled heme-iron. Each day, reticuloendothelial system (RES) macrophages recycle heme-iron by ingesting almost 5 million senescent or damaged RBCs per second; the bone marrow utilizes this recycled iron to produce new RBCs. As a consequence genetic defects in macrophage iron recycling result in anemia. Despite the importance of heme in iron recycling, the pathways responsible for heme transport and trafficking in RES macrophages remain poorly understood. After erythrophagocytosis, the iron is enzymatically released from heme by heme oxygenases (HMOX1) in the cytosol. This iron can either be stored in ferritin (FTH1) or exported out of the cell by ferroportin (FPN1) to be reutilized for new RBC production in the bone marrow. Consequently, genetic disruption in HMOX1, FPN1, and FTH1 ? steps in the heme-iron recycling pathway ? causes embryonic lethality in mice. We identified the first eukaryotic heme importer/transporter, HRG1 and showed that it is essential for transporting heme from the macrophage phagolysosome into the cytoplasm in macrophages. Our recent studies show that HRG1-deficient mice are viable even though they are incapable of recycling heme-iron and accumulate 10-fold excess heme within macrophages. The mice are tolerant to heme toxicity because they sequester heme inside lysosomes, which become 10-100 times larger than normal, by forming hemozoin - large multimeric heme crystals heretofore only identified in blood-feeding organisms to detoxify heme. Heme tolerance requires a fully-operational heme degradation pathway as haploinsufficiency of HMOX1 combined with HRG1 deficiency causes perinatal lethality, demonstrating a synthetic lethal interaction. Our exciting results suggest the existence of a previously unanticipated pathway for heme detoxification and tolerance in mammals. The studies in this proposal are designed to uncover the molecular basis of HRG1-mediated heme tolerance. We will test the hypotheses that heme tolerance is conferred by the coordinated regulation of heme transport by HRG1 and its genetic and physical interactions with heme degradation and additional components of the heme trafficking machinery. We will utilize (a) a genetic approach to elucidate the regulatory mechanisms of hemozoin formation and heme tolerance; and (b) a cell biological approach to identify the mechanisms for the synthetic lethal interactions between HRG1 and HMOX1. Our goals are to acquire a deep understanding for the molecular basis for heme trafficking and recycling and its role in heme tolerance in mammals.